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PEX11 promotes peroxisome division independently of peroxisome metabolism

 

Xiaoling Li and Stephen J. Gould

Department of Biological Chemistry, The John Hopkins University School of Medicine, Baltimore, MD 21205

 

Review by Nicole Hesson

 

Summary and Critique

            Peroxisomes are membrane bound organelles that import all of their protein, and are important in many different lipid metabolism, especially fatty acid β-oxidation.  Studies have shown that PEX11 may be involved in peroxisome metabolism or division.  PEX11 in humans has at least two forms, PEX11α and PEX11β.  There are both known to be integral membrane proteins, and the β form has been shown to increase the number of peroxisomes in a cell.  For this reason, the authors choose to focus on PEX11β rather than both components.

In this paper, Li and Gould are trying to prove that the PEX11 gene is responsible for “peroxisome division independent of peroxisome metabolism.”  First, they performed a test to determine if PEX11 is sufficient for division without growth stimulus.  They grew the cells in media lacking any growth promoters and overexpressed different peroxisome genes.  PEX11 was the only gene that initiated peroxisome division.  The second hypothesis they tested was whether or not the effect of PEX11β in the former experiment was dependent on normal peroxisome metabolism.  They compared the growth of normal fibroblast cells and cells lacking the PEX5 gene.  PEX5-/- cells do not perform normal metabolic functions because they do not transcribe mRNA.  Therefore, the cells do not import proteins into the lumen, nor do they β-oxidize fatty acids or synthesize ether lipids.  The over expression of PEX11β had an increasing effect on the number of peroxisomes in both normal and defective cells.  They then tested if the PEX11β gene was dependent on the media the cells were grown on.  On surroundings lacking β-oxidation substrates, wild type cells contained twice as many peroxisomes as the PEX11β-/- cells, revealing that “PEX11 proteins promote division regardless of the metabolic state of the peroxisomes” (Li and Gould, 2002).   

            Figure 1: Cells in panels A, C, and E have been microinjected with PEX11βmyc.  These cells have been induced to over express PEX11.  Control cells in panels B, D, and F have been microinjected with PMP34myc.  PMP34 is a human homologue of another yeast protein needed for β-oxidation.  Antibodies for the myc tag as well as PEX14 were used to detect indirect immunoflourescence.  PEX14 is a protein normally found in peroxisome membranes.  Cells in A and B were incubated for 1.5 hours, C and D for 4.5 hours, and E and F for 48 hours.  At 1.5 hours, peroxisome level is normal in both sets of cells.  After 4.5 hours, there appear to be more peroxisomes in the PEX11β cells.  After 48 hours, it is clear that PEX11β has had an effect on the experimental cells.  The PMP34 cells showed no significant increase in abundance of peroxisomes.  This figure is telling us that proliferation of peroxisomes does not happen immediately.  It takes at least 4.5 hours to show any significant difference.  This figure does a good job of proving to the reader what it is meant to prove.

            Figure 2: Normal cells in B and C have been transfected with PMP34myc, while cells in D and E have been transfected with PEX11βmyc.  Antibodies for the myc tag were used in panels B and D, while antibodies for PEX14 were used in C and E.  Other PMP genes were also transfected, but they showed the same results as PMP34.  Panel A simply quantifies the data shown in B-E, that cells induced with PEX11β had a higher number of peroxisomes.  Figure 2 as a whole drives home the fact that overexpression of any PMP is not responsible for an increase in peroxisome, but it is in fact the over expression of PEX11β that is causing the increase.  While this data does show that, it would have been nice to see either immunoflourescence pictures or graph bars, or both, displaying the other PMP genes that had been tested.  This would have been more convincing to prove that PEX11β is the gene responsible.  Just showing one PMP example is not enough.  Several are needed.

            Figure 3:  Cells in B and C have been transfected with PMP34myc, while cells in D and E have been transfected with PEX11βmyc.  Antibodies for the myc tag were used in panels B and D, while antibodies for PEX14 were used in C and E.  The cells in this figure were not normal cells.  The peroxisomes in these cells lacked the PEX5 gene.  The PEX5 gene is an important gene for normal peroxisome metabolism.  See above for details on PEX5 mutants.  In PEX5 mutants overexpressing the PEX11β gene, the number of peroxisomes was much higher than untransfected cells (first bar in A), as well as those cells transfected with PMP34myc (bar 2).  PEX11β cells are represented in bar 3.  This figure tells us that PEX11β can still promote peroxisome division even if peroxisome metabolic actions are non functional – division and metabolism are independent of one another.  Once again, it would have been good to see more PMP genes.  It also would have been nice to see mutants other than the PEX5 gene.  However, this figure is still fairly convincing.     

            Figure 4: Normal lab strain yeast cells were made to express GFP/PTS1, which causes GFP to be imported into the peroxisome lumen.  Panels A and B show the difference in GFP containing peroxisomes when grown on different media.  A was grown on glucose, a sugar, and B shows cells grown on oleic acid, a fatty acid.  Since peroxisomes are the sole site of fatty acid oxidation in yeast cells, peroxisome number should increase on a fatty acid, as shown in panel B.  Cells in A and B contain plasmids with theGAL1 promoter.  Panels C and D use a different strain of yeast.  These cells have had the PEX11 gene deleted, contain GFP/PTS1, and contain a high copy GAL1 promoter plasmid, but the vectors do not induce any genes to be expressed.  Cells in C were grown on a glucose media, and cells in D were grown on a galactose media.  There appeared to be more peroxisomes in panel D, and this was due to the removal from the glucose repression.  Neither glucose nor galactose, both sugars, showed as much peroxisome abundance as the oleic acid in panel B.  The first four panels are control panels.  Panel E included cells with gal-induced expression of the PEX13 gene, and panel F included cells with gal-induced expression of the YPR128C gene.  PEX13 is involved in protein transport, and YPR128C carries adenine to the membrane of the peroxisome.  Both are PMPs.  Neither E nor F displayed any more peroxisome quantity than panel D.  This data tells us that more than PMP overexpression is necessary to increase peroxisome division.  Panel G cells are similar to cells in panels E and F, except the plasmid has been constructed to overexpress PEX11.  These cells show peroxisome numbers close to those seen in panel B.  Since the cells were not grown on a fatty acid media, this data suggests that PEX11 is the cause of the peroxisome increase.  Panels H and I show cells with the PEX11 gene deleted, GFP/PTS1 added, a high copy GAL1 promoter plasmid, and the POX1 gene deleted.  This gene is the first step in the β-oxidation pathway, and is essential for yeast cells to oxidize any fatty acids.  The GAL1 plasmid in panel H induced expression of PEX13, while the plasmid in panel I induced expression of PEX11.  Panel H was no different that panels E or F, while panel I exhibits peroxisome levels similar to those seen in panels G and B.  Once more, this data shows us that PEX11 is sufficient to increase the number of peroxisomes in a cell.  I did not really understand this figure at first, but now I think it is very nice.  They tested yeast strains with one mutation, strains with more than one mutation, vectors with no genes, vectors built to express different peroxisome genes, and strains grown on different growth mediums.  All data show us that PEX11β is the gene that is causing the increase in peroxisome number, regardless of number of mutations or growth media.     

            Figure 5: Panels B and C contain wild type mouse fibroblasts, and panels D and E contain PEX11β-/- cells.  Both were grown on a normal media as well as a serum free media, which contained no lipids or substrates for the oxidation path.  B and D were used antibodies to PEX14, and C and E used antibodies to a matrix marker enzyme catalase (? I assume this is a protein in the cell.).  If PEX11β plays a role in oxidation, the number of peroxisomes should be the same.  If the lipids and substrates are missing, the level of peroxisomes in wild type and mutant should be low.  If the gene is responsible for division of peroxisomes, there ought to be a significant difference in the levels of peroxisomes.  PEX11β will increase the number of peroxisomes regardless of the media.  This is exactly what we see.  The wild type cells show about twice as many peroxisomes as the mutant cells.  It has been determined that PEX11β functions to promote peroxisome division.

            Overall, the paper was well done.  It was well written, and for the most part the figures were convincing to the reader.

 

Future Experiments

            We now know that the PEX11β gene is involved in peroxisome division, but it is still not known how the proteins made from the gene promote division.  How do they interact with other proteins?  Li and Gould suggest that perhaps the level of peroxisome division is dependent on how much PEX11 is concentrated in the membrane.  To test this, it would be necessary to test different concentrations of PEX11 in the membrane.  The control would be a wild type cell with normally functioning peroxisomes.  A negative control would be a PEX11 deficient mutant with no PEX11 proteins in the membrane.  It would be possible to overexpress PEX11 at different concentration levels in order to test the hypothesis.  This could be tested using the same procedures the writers used in this paper – transfection, microinjection, immunoflourescence and multiple antibodies.

The authors also suggest that PEX11 proteins are affected by modification after translation.  In order to determine if this is the case, researchers would simply have to perform column chromatography.  If PEX11 proteins are modified post-translationally, the protein from the cell would be a larger molecular weight than if the protein was made in the lab from the mRNA strand.  They could also do something similar to what we did in lab.  The could make a protein, and if it is nonfunctional, it may be deduced that perhaps modification takes place in the cell after translation.  It is possible that the PEX11 proteins either modify lipids directly or act to bring other proteins that do modify lipids to the membrane of the peroxisome.  It may be possible to establish if the other proteins need to bind to the PEX11 proteins in order to work.  A nickel column could determine this.  Cooperative proteins bound together will have a higher weight than just a PEX11 protein alone or another protein alone.      

The authors also suggest that the loss of PEX11 proteins may physically change the membrane of the peroxisome.  A change in the membrane could affect many types of transmembrane transport systems.  This could be determined by removing PEX11 proteins from a wild type cell and doing FRAP to see how lipids and proteins are being transported.  A control would be a regular wild type cell, where proteins and lipids move freely.  Another would be a PEX11-/- mutant.  This would be a negative control.  Removal of PEX11 proteins should look like this data.  If a physical change does occur, it would be possible to see a change in the shape of the peroxisome.

 

 

 

References

 

Li, X., and S. Gould. 2002. PEX11 promotes peroxisome division independently of peroxisome metabolism. J. Cell

Biol.156: 643-651

 

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